Labeled Biomolecular Compositions and Methods for the Production and Uses Thereof

ABSTRACT

Disclosed herein is a set of  E. coli  single-protein production (SPP) technologies with protein NMR (SPP-NMR) for (i) using isotope-enriched membrane proteins produced with the SPP system in screening detergent conditions suitable for purification and/or three-dimensional structure analysis without the requirement for protein purification, (ii) producing  2 H,  13 C,  15 N enriched proteins suitable for high throughput and membrane protein NMR studies, and (iii) labeling with  13 C- 15 N specific peptide bonds in proteins (referred to herein as SPP-PBL).

CLAIM OF PRIORITY

This application claims priority to U.S. Provisional Application No.60/918,418, filed Mar. 16, 2007, the disclosure of which is herebyincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NIH Grant NumbersU54 GMO74958 and U54 GMO75026.

STATEMENT REGARDING REFERENCES

All patents, publications and non-patent references referred to hereinshall be considered incorporated by reference into this application intheir entireties.

BACKGROUND OF THE INVENTION

Determination of precise and accurate protein structures by NuclearMagnetic Resonance (hereinafter “NMR”) generally requires weeks or evenmonths to acquire and interpret all of the necessary NMR data,particularly for completing side chain resonance assignments. However,medium-accuracy fold information can often provide important and helpfulclues about protein evolution and biochemical function(s). A largelyautomatic strategy for rapid determination of medium-accuracy proteinbackbone structures has been previously described (Zheng, D., Huang, Y.J., Moseley, H. N. B., Xiao, R., Aramini, J., Swapna, G. V. T.;Montelione, G. T. (2003) Automated protein fold determination using aminimal NMR constraint strategy. Protein Science 2003, 12: 1232-1246.).This strategy of rapid fold determination derives from ideas originallyintroduced for determining medium-accuracy NMR structures of largeproteins (Gardner, K. H., Rosen, M. K., and Kay, L. E. (1997). Globalfolds of highly deuterated, methyl-protonated proteins bymultidimensional NMR. Biochemistry 36, 1389-1401.), using deuterated,¹³C-, ¹⁵N-enriched protein samples with selective protonation ofsidechain methyl groups (¹³CH₃). Data collection includes acquiring NMRspectra suitable for automatic analysis of assignments for backbone andsidechain ¹⁵N, H^(N) resonances, and sidechain ¹³CH₃ methyl resonances.In some cases, assignments are also determined for ¹H and/or ¹³C atomsof labeled Tyr and Phe residues. NMR resonance assignments can bedetermined by automated NMR assignment programs, such as the programAutoAssign (Moseley, H. N., Monleon, D., and Montelione, G. T. (2001).Automatic determination of protein backbone resonance assignments fromtriple resonance nuclear magnetic resonance data. Methods Enzymol 339,91-108; Moseley, H. N., and Montelione, G. T. (1999). Automated analysisof NMR assignments and structures for proteins. Curr Opin Struct Biol 9,635-642; Zimmerman, D. E., Kulikowski, C. A., Huang, Y.; Feng, W.,Tashiro, M., Shimotakahara, S., Chien, C., Powers, R., and Montelione,G. T. (1997). Automated analysis of protein NMR assignments usingmethods from artificial intelligence. J Mol Biol 269, 592-610;Zimmerman, D. E., and Montelione, G. T. (1995). Automated analysis ofnuclear magnetic resonance assignments for proteins. Curr Opin StructBiol 5, 664-673). Three-dimensional structures can be analyzedautomatically with programs like AutoStructure (Huang, Y. J., Moseley,H. N., Baran, M. C., Arrowsmith, C., Powers, R., Tejero, R., Szyperski,T., and Montelione, G. T. (2005). An integrated platform for automatedanalysis of protein NMR structures. Methods Enzymol 394, 111-141; Huang,Y. J., Tejero, R., Powers, R., and Montelione, G. T. (2006). Atopology-constrained distance network algorithm for protein structuredetermination from NOESY data. Proteins 62, 587-603). The total timerequired for collecting and processing NMR spectra for themedium-accuracy strategy can be relatively short. For example, using NMRdata on ²H, ¹³C, ¹⁵N-enriched proteins with protonated methyl (and/oraromatic) groups, published NMR software packages like AutoAssign andAutoStructure can be used to process NMR spectra, carry out resonanceassignments, interpret Nuclear Overhauser Enhancement Spectroscopy(hereinafter “NOESY”) data, and generate medium-accuracy structureswithin a few days. These structures provide essential three-dimensionalinformation for characterizing biological activities of proteins, andare good starting points for further refinement to high precision andaccuracy using additional NMR data. The feasibility of this combineddata collection and analysis strategy starting from raw NMR time domaindata has already been demonstrated by automatic analysis of a mediumaccuracy structure of the Z domain of Staphylococcal protein A (Zheng,D., Huang, Y. J., Moseley, H. N. B., Xiao, R., Aramini, J., Swapna, G.V. T.; Montelione, G. T. (2003) Automated protein fold determinationusing a minimal NMR constraint strategy. Protein Science 2003, 12:1232-1246).

Perdeuteration (the enrichment of proteins with ²H) is also aprerequisite for using some of the most advanced NMR methods forstudying the three-dimensional structures of membrane proteins by NMR(Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001).Structure of outer membrane protein A transmembrane domain by NMRspectroscopy. Nat Struct Biol 8, 334-338; Fernandez, C., Hilly, C.,Wider, G., Guntert, P., and Wuthrich, K. (2004). NMR structure of theintegral membrane protein OmpX. J Mol Biol 336, 1211-1221; Fernandez,C., Hilty, C., Wider, G., and Wuthrich, K. (2002). Lipid-proteininteractions in DHPC micelles containing the integral membrane proteinOmpX investigated by NMR spectroscopy. Proc Natl Acad Sci USA 99,13533-13537; Fernandez, C., and Wuthrich, K. (2003). NMR solutionstructure determination of membrane proteins reconstituted in detergentmicelles. FEBS Lett 555, 144-150; Sorgen, P. L., Cahill, S. M.,Krueger-Koplin, R. D., Krueger-Koplin, S. T., Schenck, C. C., andGirvin, M. E. (2002a). Structure of the Rhodobacter sphaeroideslight-harvesting 1 beta subunit in detergent micelles. Biochemistry 41,31-41; Sorgen, P. L., Hu, Y., Guan, L., Kaback, H. R., and Girvin, M. E.(2002b). An approach to membrane protein structure without crystals.Proc Natl Acad Sci USA 99, 14037-14040; Tamm, L. K., Abildgaard, F.,Arora, A., Blad, H., and Bushweller, J. H. (2003). Structure, dynamicsand function of the outer membrane protein A (OmpA) and influenzahemagglutinin fusion domain in detergent micelles by solution NMR. FEBSLett 555, 139-143)). These methods require use of ²H, ¹³C, ¹⁵N-enrichedmembrane protein samples with ¹³C-¹H (or ¹²C-¹H) methyl labels.Production of such samples can be expensive ($1,500-$10,000 per sample),limiting the applicability of this approach. The high cost of sampleproduction greatly limits the applicability of powerful automatedstructure analysis methods (Zheng, D., Huang, Y. J., Moseley, H. N. B.,Xiao, R., Aramini, J., Swapna, G. V. T.; Montelione, G. T. (2003)Automated protein fold determination using a minimal NMR constraintstrategy. Protein Science 2003, 12: 1232-1246.) and certain powerfulmembrane protein structure analysis methods (Arora, A., Abildgaard, F.,Bushweller, J. H., and Tamm, L. K. (2001). Structure of outer membraneprotein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8,334-338; Fernandez, C., Hilty, C., Wider, G., Guntert, P., and Wuthrich,K. (2004). NMR structure of the integral membrane protein OmpX. J MolBiol 336, 1211-1221; Fernandez, C., Hilty, C., Wider, G., and Wuthrich,K. (2002). Lipid-protein interactions in DHPC micelles containing theintegral membrane protein OmpX investigated by NMR spectroscopy. ProcNatl Acad Sci USA 99, 13533-13537; Fernandez, C., and Wuthrich, K.(2003). NMR solution structure determination of membrane proteinsreconstituted in detergent micelles. FEBS Lett 555, 144-150; Sorgen, P.L., Cahill, S. M., Krueger-Koplin, R. D., Krueger-Koplin, S. T.,Schenck, C. C., and Girvin, M. E. (2002a). Structure of the Rhodobactersphaeroides light-harvesting 1 beta subunit in detergent micelles.Biochemistry 41, 31-41; Sorgen, P. L., Hu, Y., Guan, L., Kaback, H. R.,and Girvin, M. E. (2002b). An approach to membrane protein structurewithout crystals. Proc Natl Acad Sci USA 99, 14037-14040; Tamm, L. K.,Abildgaard, F., Arora, A., Blad, H., and Bushweller, J. H. (2003).Structure, dynamics and function of the outer membrane protein A (OmpA)and influenza hemagglutinin fusion domain in detergent micelles bysolution NMR. FEBS Lett 555, 139-143)).

There are three principle approaches for membrane protein structureanalysis by NMR. The first approach is solution-state NMR, which can beused to determine three-dimensional structures of detergent-solubilizedmembrane proteins using conventional triple-resonance NMR methods withsensitivity-enhanced Transverse Relaxation Optimized Spectroscopy(hereinafter “TROSY”) detection methods (Arora, A., Abildgaard, F.;Bushweller, J. H., and Tamm, L. K. (2001). Structure of outer membraneprotein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8,334-338; Fernandez, C., Hilty, C., Wider, G., Guntert, P., and Wuthrich,K. (2004). NMR structure of the integral membrane protein OmpX. J MolBiol 336, 1211-1221; Fernandez, C., Hilty, C., Wider, G., and Wuthrich,K. (2002). Lipid-protein interactions in DHPC micelles containing theintegral membrane protein OmpX investigated by NMR spectroscopy. ProcNatl Acad Sci USA 99, 13533-13537; Fernandez, C., and Wuthrich, K.(2003). NMR solution structure determination of membrane proteinsreconstituted in detergent micelles. FEBS Left 555, 144-150; Sorgen, P.L., Cahill, S. M., Krueger-Koplin, R. D., Krueger-Koplin, S. T.,Schenck, C. C., and Girvin, M. E. (2002a). Structure of the Rhodobactersphaeroides light-harvesting 1 beta subunit in detergent micelles.Biochemistry 41, 31-41; Sorgen, P. L., Hu, Y., Guan, L., Kaback, H. R.,and Girvin, M. E. (2002b). An approach to membrane protein structurewithout crystals. Proc Natl Acad Sci USA 99, 14037-14040; Tamm, L. K.,Abildgaard, F., Arora, A., Blad, H., and Bushweller, J. H. (2003).Structure, dynamics and function of the outer membrane protein A (OmpA)and influenza hemagglutinin fusion domain in detergent micelles bysolution NMR. FEBS Lett 555, 139-143). The methods under this approachrequire the use of ²H, ¹³C, ¹⁵N-enriched membrane protein samples with¹³C-¹H methyl labels.

The other two approaches are two methods of solid-state NMR, which havebeen successfully applied to membrane protein structure analysis. One ofthese approaches, Oriented Solid-State NMR, uses molecular orientationto overcome the line-broadening effects of dipolar coupling and chemicalshift that otherwise complicate solid-state NMR spectra of proteins.Pioneered for applications to membrane proteins, samples of lipidbilayers or bicelles are statically-oriented in a special NMR probe,providing a high resolution NMR spectrum that includes information aboutinteratomic bond orientations. This approach, while still underdevelopment, has already been used to determine three-dimensionalstructures of small membrane proteins in lipid bilayers (De Angelis, A.A., Howell, S. C., Nevzorov, A. A., and Opella, S. J. (2006). Structuredetermination of a membrane protein with two trans-membrane helices inaligned phospholipid bicelles by solid-state NMR spectroscopy. J Am ChemSoc 128, 12256-12267; Kim; S., Quine, J. R., and Cross, T. A. (2001).Complete cross-validation and R-factor calculation of a solid-state NMRderived structure. J Am Chem Soc 123, 7292-7298; Marassi, F. M., andOpella, S. J. (2002). Using pisa pies to resolve ambiguities in angularconstraints from PISEMA spectra of aligned proteins. J Biomol NMR 23,239-242; Marassi, F. M., and Opella, S. J. (2003). Simultaneousassignment and structure determination of a membrane protein from NMRorientational restraints. Protein Sci 12, 403-411; Opella, S. J. (2003).Membrane protein NMR studies. Methods Mol Biol 227, 307-320; Park, S.H., Mrse, A. A., Nevzorov, A. A., Mesleh, M. F., Oblatt-Montal, M.,Montal, M., and Opella, S. J. (2003). Three-dimensional structure of thechannel-forming trans-membrane domain of virus protein “u” (Vpu) fromHIV-1. J Mol Biol 333, 409-424; Valentine, K. G., Mesleh, M. F., Opella,S. J., Ikura, M., and Ames, J. B. (2003). Structure, topology, anddynamics of myristoylated recoverin bound to phospholipid bilayers.Biochemistry 42, 6333-6340; Zeri, A. C., Mesleh, M. F., Nevzorov, A. A.,and Opella, S. J. (2003). Structure of the coat protein in fdfilamentous bacteriophage particles determined by solid-state NMRspectroscopy. Proc Natl Acad Sci USA 100, 6458-6463).

The second solid-state NMR approach, Magic Angle Spinning (hereinafter“MAS”) NMR, provides another method for narrowing the broad lines ofsolid-state NMR samples by minimizing the effects of dipolar coupling byrapidly spinning the solid sample at a special orientation) (54.7°relative to the applied magnetic field. MAS methods have been furtherdeveloped to the point where it is now possible to obtain completeresonance assignments and three-dimensional structures of small proteinsin the solid state, including membrane proteins (Astrof, N. S., Lyon, C.E., and Griffin, R. G. (2001). Triple resonance solid state NMRexperiments with reduced dimensionality evolution periods. J Magn Reson152, 303-307; Castellani, F., van Rossum, B., Diehl, A., Schubert, M.,Rehbein, K., and Oschkinat, H. (2002). Structure of a protein determinedby solid-state magicangle-spinning NMR spectroscopy. Nature 420, 98-102;Castellani, F., van Rossum, B. J., Diehl, A., Rehbein, K., andOschkinat, H. (2003). Determination of solid-state NMR structures ofproteins by means of three-dimensional 15N-13C-13C dipolar correlationspectroscopy and chemical shift analysis. Biochemistry 42, 11476-11483;Igumenova, T. I., McDermott, A. E. Zilm, K. W., Martin, R. W., Paulson,E. K., and Wand, A. J. (2004a). Assignments of carbon NMR resonances formicrocrystalline ubiquitin. J Am Chem Soc 126, 6720-6727; Igumenova, T.I., Wand, A. J., and McDermott, A. E. (2004b). Assignment of thebackbone resonances for microcrystalline ubiquitin. J Am Chem Soc 126,5323-5331; Jaroniec, C. P., MacPhee, C. E., Bajaj, V. S., McMahon, M.T., Dobson, C. M., and Griffin, R. G. (2004). High-resolution molecularstructure of a peptide in an amyloid fibril determined by magic anglespinning NMR spectroscopy. Proc Natl Acad Sci USA 101, 711-716; Krabben,L., van Rossum, B. J., Castellani, F., Bocharov, E., Schulga, A. A.,Arseniev, A. S., Weise, C., Hucho, F., and Oschkinat, H. (2004). Towardsstructure determination of neurotoxin II bound to nicotinicacetylcholine receptor: a solid-state NMR approach. FEBS Left 564,319-324; Petkova, A. T., Baldus, M., Belenky, M., Hong, M., Griffin, R.G., and Herzfeld, J. (2003). Backbone and side chain assignmentstrategies for multiply labeled membrane peptides and proteins in thesolid state. J Magn Reson 160, 1-12; Rienstra, C. M., Tucker-Kellogg,L., Jaroniec, C. P. Hohwy, M., Reif, B., McMahon, M. T., Tidal., B.,Lozano-Perez, T., and Griffin, R. G. (2002). De novo determination ofpeptide structure with solid-state magic-angle spinning NMRspectroscopy. Proc Natl Acad Sci USA 99, 10260-10265).

Solid-state NMR has tremendous potential for providing three-dimensionalstructures of many membrane proteins that cannot be crystallized.Oriented Solid-State NMR experiments are particularly well-suited fordetermining structures of helical membrane proteins, and MASexperiments, which can identify dipolar interactions between backboneatoms in adjacent beta strands, are especially well-suited for beta-typemembrane structures, though it may also be possible to determinestructures of alpha-helical proteins with these new methods.

NMR has special value in structural genomics efforts for rapidlycharacterizing the “foldedness” of specific protein constructs (Kennedy,M. A., Montelione, G. T., Arrowsmith, C. H., and Markley, J. L. (2002).Role for NMR in structural genomics. J Struct Funct Genomics 2, 155-169;Montelione, G. T. (2001). Structural genomics: an approach to theprotein folding problem. Proc Natl Acad Sci USA 98, 13488-13489). Thedispersion and line shapes of resonances measured in one-dimensionalH-NMR and two-dimensional N—H or C—H correlation spectra provide“foldedness” criteria with which to define constructs and solutionconditions that provide folded protein samples (see FIG. 1). Therequired isotopic enrichment with ¹⁵N is relatively inexpensive, and thetwo-dimensional ¹³N-¹H correlation spectra can be recorded in tens ofminutes with conventional NMR systems.

An E. coli Single Protein Production (hereinafter “SPP”) bacterialexpression system has been previously described that utilizes acombination of attributes cold-inducible promoters, low temperature,induction of the mRNA-specific endoribonuclease MazF causing host cellgrowth arrest, and culture condensation to facilitate stable, high levelprotein expression (almost 30% of total cellular protein) withoutbackground protein synthesis (Suzuki, M., Roy, R., Zheng, H., Woychik,N., and Inouye, M. (2006). Bacterial bioreactors for high yieldproduction of recombinant protein. J Biol Chem 281, 37559-37565; Suzuki,M., Zhang, J., Liu, M., Woychik, N. A., and Inouye, M. (2005). Singleprotein production in living cells facilitated by an mRNA interferase.Mol Cell 18, 253-261). This expression system has been shown to providespecific labeling with selenomethionine and fluorophenylalanine (Suzuki,M., Roy, R., Zheng, H., Woychik, N., and Inouye, M. (2006). Bacterialbioreactors for high yield production of recombinant protein. J BiolChem 281, 37559-37565). Moreover, using an optimized SPP vector,exponentially growing cultures can be condensed 40-fold withoutsignificantly affecting protein yields (Suzuki, M., Roy, R., Zheng, H.,Woychik, N., and Inouye, M. (2006). Bacterial bioreactors for high yieldproduction of recombinant protein. J Biol Chem 281, 37559-37565). Thishas the potential to lower sample labeling costs to a small percentageof the cost of traditional isotope-labeling experiments.

The compositions, systems, and methods of the present invention provideeffective means to screen conditions for membrane protein purification,membrane protein structure analysis, and to determine three-dimensionalprotein structures using deuterium-decoupled NMR methods suitable forrapid structure analysis and analysis of large protein structures. Thepresent invention is also advantageous in that it provides means forproduction of deuterated protein samples at reduced cost.

SUMMARY OF THE INVENTION

Disclosed herein are novel processes for (i) producing ²H, ¹³C, ¹⁵Nenriched proteins using an E. coli SPP-NMR expression system (as furtherdescribed herein), (ii) using isotope-enriched membrane proteinsproduced with an SPP-NMR system for screening detergent conditionssuitable for purification and/or three-dimensional structure analysis,and (iii) labeling with ¹³C-¹⁵N specific peptide bonds in proteins(further described and referred to herein as SPP-PBL). The methods ofisotope-enrichment can be combined with high throughput data collectionand analysis methods to provide rapid analysis of small proteinstructures by NMR. Conditions identified by detergent screening can beused for purifying membrane proteins or for direct determination oftheir three-dimensional structures by NMR without purification.

In certain embodiments, the present invention is directed tocompositions, systems, and methods for producing ²H, ¹³C, ¹⁵N enrichedproteins using an SPP-NMR system, methods for using theseisotope-enriched proteins for screening detergent conditions suitablefor purification and/or three-dimensional structure analysis, andlabeling with ¹³C-¹⁵N specific peptide bonds in proteins.

In other embodiments, the present invention is directed to an SPP-NMRsystem capable of separately inducing an mRNA-specific endoribonucleaseand a target protein.

In further embodiments, the present invention is directed to a method ofinducing a target protein by first contacting a vector containing a geneencoding an mRNA-specific endoribonuclease and a gene encoding a targetprotein with a composition suitable for inducing the mRNA-specificendoribonuclease, and then contacting the same vector with a compositionsuitable for inducing the target protein.

In other embodiments, the present invention is directed to a method ofinducing a target protein by first contacting a vector containing a geneencoding an mRNA-specific endoribonuclease and a gene encoding a targetprotein with tetracycline to induce the mRNA-specific endoribonuclease,then eliminating background protein, then contacting the vector with anisotope-enriched media for a sufficient time to acclimate the vector tothe media, and finally contacting the vector with IPTG to induce thetarget protein.

In further embodiments, the present invention is directed to a method ofproducing an isotope-labeled protein by first contacting a vectorcontaining a gene encoding an mRNA-specific endoribonuclease and a geneencoding a target protein with a composition suitable to induce themRNA-specific endoribonuclease, then contacting the vector with aselected isotope-labeled media to label the target protein, and finallycontacting the vector with a composition suitable for inducing thetarget protein to produce an isotope-labeled protein.

In other embodiments, the present invention is directed toward a novelvector that contains the promoter-operator region of the tetA (tet^(P0))and tetR genes from Tn10 and a Multiple Cloning Site downstream of thetetA^(P0), allowing two proteins to be induced independently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of ¹⁵N-¹H HSQC correlation spectra fordisordered and well-folded proteins. (A) HSQC spectrum of T.thermophilus BRCT domain of the DNA ligase, a homologue of NESG targetWR64, which has a well-defined three-dimensional structure in aqueoussolution. (B) HSQC spectrum of D. melanogaster Par 1 C-terminal domain,a domain construct, which is predominantly disordered under theconditions of these measurements.

FIG. 2 depicts NMR methods for protein structure analysis withoutpurification of isotope-enriched proteins produced with the SPP system.

FIG. 3 depicts ¹⁵N-¹H HSQC spectra measured with non-purified,¹⁵N-labeled RAMP-4 in (A) 10% (vol/vol) bddm (B) 5% (vol/vol) bddm (C)10% (vol/vol) Idao (D) 10% (vol/vol) zwittergent and (E) 10% DPC(vol/vol) micelles.

FIG. 4. depicts ¹⁵N-¹H TROSY spectra of non-purified, ¹⁵N-labeled RAMP-4in (A) 10% (vol/vol) bddm (B) 5% (vol/vol) bddm (C) 10% (vol/vol) Idao(D) 10% (vol/vol) zwittergent and (E) 10% DPC (vol/vol) micelles.

DETAILED DESCRIPTION OF THE INVENTION

The claimed invention introduces a new technology that makes the SPPsystem more suitable for isotope-enrichment and perdeuteration ofproteins. The claimed Single Protein Production-Nuclear MagneticResonance (hereinafter “SPP-NMR”) system utilizes a clonedpromoter-operator region of the tetA (tetA^(P0)) and tetR genes fromTn10. It is known that the expression of the tetA gene is repressed byTetR (tet repressor) and is regulated by tetracycline. Since theMultiple Cloning Site (hereinafter “MCS”) exists downstream oftetA^(P0), any gene of interest can be cloned into this plasmid.Therefore, expression of the gene cloned into the MCS is induced by theaddition of tetracycline. The unique feature of the SPP-NMR system isthat the mRNA-specific endoribonuclease and the target protein can beinduced independently. In one embodiment of the claimed SPP-NMR system,the mazF gene is cloned into this tetracycline inducible vector, so thatMazF and the target protein are induced by the addition of tetracyclineand IPTG, respectively. However one skilled in the art would recognizethat any mRNA-specific endoribonuclease gene could be substituted forthe maze gene in this embodiment. In contrast to the previousco-inducible system, the use of the new vector allows one to induce MazFor any other mRNA-specific endoribonuclease first, eliminate thebackground protein production, appropriately acclimate the cells inisotope-enriched media, and then induce the target protein. In thismanner, one can now induce the production of the target protein onlyafter the medium is exchanged with a specific medium for isotopelabeling. This is an important new innovation that is essential for thepractical production of perdeuterated membrane protein samples for NMRstudies.

Protein Perdeuteration Using the SPP-NMR System

The high cost of obtaining ²H, ¹³C, ¹⁵N-enriched protein samples with¹H-¹³C and/or ¹H-¹²C labeled methyls and/or aromatic sidechains remainsa major bottleneck in the application of various NMR methods. TheSPP-NMR System, which combines the SPP expression system with isotopelabeling protocols, provides a powerful method to obtain NMR qualitysamples at a drastically reduced cost. A unique feature of the SPP-NMRSystem is the ability to not only grow cells in the absence ofdeuterium, but to condense bacteria cultures up to forty fold (40×)prior to the introduction of deuterium, isotope enriched media (Suzuki,M., Roy, R., Zheng, H., Woychik, N., and Inouye, M. (2006). Bacterialbioreactors for high yield production of recombinant protein. J BiolChem 281, 37559-37565). This application can be applied to a variety ofinduction systems, including tetracycline and IPTG inducible systems.Condensation limits may vary depending on the protein of interest andtherefore optimal fold-condensation needs to be evaluated individuallyfor each protein target. Cultures are condensed on a small scale at 5×,10×, 20×, 30×, and 40× and compared to 1× cultures. The highest foldcondensation showing equal protein expression levels as IX control isthen chosen as optimal for labeling experiments.

The SPP-NMR method allows for soluble bacterial cell lysates to beanalyzed directly or with minimal purification steps. Such analysis ispossible since the only protein expressed, and therefore the onlyprotein labeled following introduction of NMR isotopes, is the targetprotein. Once resuspended in a reduced volume, the protein of interestis expressed in a condensed state with no loss in overall yield. Thisresults in a 5× to 40× cost reduction directly translating into a samplepreparation cost reduction from $1500-$10,000 for traditional methods to$37.5-$250 when condensed 40× with the SPP-NMR method. The use of thistechnique is widely applicable to NMR spectroscopy allowing for theproduction of highly labeled samples suitable for obtaining rapidresonance assignments at a fraction of the cost.

When utilizing the SPP-NMR method for ²H labeling, cells are initiallygrown to an OD₆₀₀ between 0.5 and 0.6 in a defined minimal media such asM9-glucose or MJ9-glucose media (Jansson, M., Li, Y. C., Jendeberg, L.,Anderson, S., Montelione, B. T., and Nilsson, B. (1996). High-levelproduction of uniformly 15N- and 13C-enriched fusion proteins inEscherichia coli. J Biomol NMR 7, 131-141). This differs fromtraditional ²H labeling methods where cells must first grow in thepresence of deuterium media to the proper cell density. Variousacclimation steps that gradually increase the percent deuterium in themedia are often necessary in order to allow cells to reach an OD₆₀₀ of0.6 in fully deuterated minimal media. With the SPP-NMR method, oncecells grown in H₂O media at 37° C. reach the proper density, they arethen shifted to lower temperature (e.g. 15° C. shaker for 45 minutes) toacclimate the culture to cold shock conditions. Cold shock conditionsfacilitate a reduction in host cellular protein expression, arequirement for single protein production and culture condensation. Boththe endoribonuclease MazF as well as the target protein are under thecontrol of an inducible cold shock promoter. Expression of MazF is theninduced with either tetracycline or IPTG for several hours (e.g., 16hours and 3 hours respectively). In a preferred embodiement, followingtetracycline-induced MazF expression and the onset of the resultingsemi-quiescent state, cell cultures are centrifuged and rinsed indeuterated minimal media to remove residual H₂0, and finally resuspendedin up to a 40× reduced volume of deuterium media containing¹³C-deuterated glucose and ¹⁵N. Specific amino acid precursors (e.g.¹³C-a-ketobutyric acid, ¹³C-a-ketoisovaleric acid,¹H-¹³C-¹⁵N-phenylalanine, and ¹H-¹³C-¹⁵N-tyrosine) are then added toallow for selective ¹H-¹³C and/or ¹H-¹²C labeling of methyls and/oraromatic sidechains. Next, cultures are typically equilibrated in theabsence of tetracycline or IPTG in the isotope enriched media for 1 hour(MazF^(IPTG)) or 4 hours (MazF^(tet)) (other times may be optimal forother specific systems) to allow for isotopes to permeate the cell priorto IPTG induction of the target protein. Finally, IPTG is added to thecondensed cell cultures, inducing the production of the single targetprotein in deuterated, isotope containing media. Target proteinproduction is allowed to proceed to a previously determined optimalexpression time, typically 20 hours. At this point cultures areharvested by centrifugation, lysed, and soluble cell extracts are eitheranalyzed directly or subsequent purification steps are carried out.

Using the SPP-NMR System to Prepare Membrane Proteins for StructureAnalysis by NMR

FIG. 2 outlines the four possible outcomes of a recombinant membraneprotein expression experiment. If the protein is expressed in arecombinant host, it may generally be classified as soluble (orpartially soluble) or insoluble (Acton, T. B., Gunsalus, K. C., Xiao,R., Ma, L. C., Aramini, J., Baran, M. C., Chiang, Y. W., Climent, T.,Cooper, B., Denissova, N. G., et al. (2005). Robotic cloning and ProteinProduction Platform of the Northeast Structural Genomics Consortium.Methods Enzymol 394, 210-243). Soluble proteins can be furtherclassified generally as “folded” or “unfolded” by using solution NMR,circular dichroism, or other methods. Most membrane proteins are“insoluble” in these simple assays; some because they are incorporateddirectly into membranes and others because they form inclusion bodies orother insoluble aggregates in the cell. The SPP-NMR System providesmeans for preparing isotope-enriched samples of these insoluble membraneproteins for structural analysis by solution state and solid state NMR.

Sample Preparation for Solution-State NMR Studies

Insoluble proteins, including those incorporated into membrane fractionsand those that form inclusion bodies or are insoluble for other reasons,may be subjected to detergent screening methods in an attempt tosolubilize them for solution-state NMR studies. An array of about 12-15micelle- and bicelle-forming detergents has been developed for this“membrane protein detergent solubilization screen” (Krueger-Koplin, R.D., Sorgen, P. L., Krueger-Koplin, S. T., Rivera-Torres, I. O., Cahill,S. M., Hicks, D. B., Grinius, L., Krulwich, T. A., and Girvin, M. E.(2004). An evaluation of detergents for NMR structural studies ofmembrane proteins. J Biomol NMR 28, 43-57). The solubilization screenwill provide a mixture of many micelle- or bicelle-solubilized proteins,of which only the target protein is isotopically-labeled, andcircumvents the necessity to purify the target proteins.Detergent-solubilization will be evaluated initially using SDS-PAGE, andthen using ¹⁵N-¹H Heteronuclear Single Quantum Coherence (hereinafter“HSQC”), ¹⁵N-¹H TROSY-HSQC, or similar kinds of NMR spectroscopy.

Robotic cloning protocols such as those described in Acton et al., 2005(Acton, T. B., Gunsalus, K. C., Xiao, R., Ma, L. C., Aramini, J., Baran,M. C., Chiang, Y. W., Climent, T., Cooper, B. Denissova, N. G., et al.(2005). Robotic cloning and Protein Production Platform of the NortheastStructural Genomics Consortium. Methods Enzymol 394, 210-243), can beapplied with the SPP-NMR labeling of membrane proteins to provide ahigh-throughput membrane protein solubilization screen. As encounteredin developing robotic methods for crystallization-screening, sometechnical challenges in reproducible pipetting of viscous detergentsolutions may be encountered. But the extensive experience in roboticCrystallization Technology Development by structural genomics groupswill be valuable in future efforts to address these issues.

Detergent solubilized proteins that provide good quality HSQC spectrawill then be prepared with ¹⁵N, ¹³C, and/or ²H enrichment, asappropriate to the size of the mixed-micelle or -bicelle system, usingthe SPP-NMR system for selective isotope enrichment. Resonanceassignments and three-dimensional structures will then be pursued withstandard TROSY-based triple-resonance NMR methods, typically using ahigh field (e.g. 800 MHz) NMR system with cryoprobe. As only thetargeted protein is isotope-enriched, it should be quite feasible tocarry out resonance assignments and complete three dimensional structuredeterminations of some membrane proteins by isotope-filtered solutionNMR methods without the need for purifying the target protein.

Although it is possible to crystallize detergent-solubilized proteins,in these mixed-micelles the solubilized protein mixture is highlyheterogeneous and not suitable for crystallization. However, thedetergent conditions screened in this way provide guidance for whichdetergents can be used for purifying the membrane protein. Thisinformation can be used to purify target proteins for crystallization,or to purify isotope-enriched target proteins for NMR studies.

Sample Preparation for Solid-State NMR Studies

Targeted ¹⁵N-enriched proteins incorporated into membrane fractions, andwithout further purification, may be prepared in tens of milligramquantities and used for MAS and Oriented solid-state NMR analysis. Thesesolid state NMR spectra will be used like solution-state NMR HSQCspectra to screen for membrane protein samples that provide good qualityNMR data. If these samples provide good quality solid-state NMR spectra,additional protein samples with ¹⁵N, ¹³C, and/or ²H isotope enrichmentshould be provided to support efforts to carry out resonance assignmentsand three dimensional structures by solid state NMR (Drechsler, A., andSeparovic, F. (2003). Solid-state NMR structure determination. IUBMBLife 55, 515-523).

Membrane proteins will be cloned and expressed in the SPP-NMR system,and then screened for soluble vs. insoluble behavior in whole cellextracts, using SDSPAGE gels to determine the fraction of expressedprotein in the whole cell, soluble extract, and membrane-only fractions.In this way, each construct will be classified as “Soluble”, “InsolubleMembrane Associated”, “Insoluble/Not-Membrane Associated”. The raresoluble membrane protein samples will then be analyzed by HSQC screeningof the whole cell extract. Insoluble protein constructs can be analyzedby two parallel pathways (illustrated in FIG. 2), utilizing eithersolid-state NMR methods or solution-state NMR methods.

Detergent Screening of Membrane Proteins that have been Isotope-Enrichedwith SPP System

The use of the SPP-NMR system for selective isotope-enrichment anddetergent screening of the ribosome-associated membrane protein 4(RAMP-4) has been demonstrated. The protein sequence is (affinitypurification tag underlined):

MNHKVHHHHHHIEGRHMAVQTPRQRLANAKFNKNNEKYRKY

GKKKEGKTEKTAPVISKTWLGILLFLLVGGGVLQLISYIL

As shown in FIGS. 3 and 4, the SPP-NMR system allows for screeningdifferent detergent conditions without purifying the ¹⁵N-enriched targetprotein to identify those which provide the best quality HSQC (FIG. 3)or TROSY-HSQC (FIG. 4) spectra. The TROSY-HSQC experiment, which hassharper line shapes for relatively slowly tumbling systems like thesemicelle-solubilized membrane proteins, is the preferred implementation.

The high quality of these TROSY-HSQC spectra suggest it will be possibleto determine complete backbone resonance assignments for membraneproteins without further purification using ¹⁵N-¹H detected ²H-decoupledtriple resonance experiments (Gardner, K. H., Rosen, M. K., and Kay, L.E. (1997). Global folds of highly deuterated, methyl-protonated proteinsby multidimensional NMR. Biochemistry 36, 1389-1401; Shan, X., Gardner,K. H., Muhandiram, D. R., Kay, L. E., and Arrowsmith, C. H. (1998).Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNAcomplex: a role for N-terminal residues in tandem binding. J Biomol NMR11, 307-318). The production of such spectra for ¹⁵N-enriched proteinsin micelles has not previously been possible. Backbone resonanceassignments obtained in this manner would provide the locations of alphahelical and beta strands in the integral membrane protein structure.

The high quality of these TROSY-HSQC spectra suggest it will be possibleto determine extensive backbone and sidechain resonance assignments formembrane proteins without further purification, using ¹⁵N-¹H-detected²H-decoupled triple resonance experiments (Gardner, K. H., Rosen, M. K.and Kay, L. E. (1997). Global folds of highly deuterated,methyl-protonated proteins by multidimensional NMR. Biochemistry 36,1389-1401; Shan, X., Gardner, K. H., Muhandirarn, D. R, Kay, L. E., andArrowsmith, C. H. (1998). Subunit-specific backbone NMR assignments of a64 kDa trp repressor/DNA complex: a role for N-terminal residues intandem binding. J Biomol NMR 11, 307-318).

The high quality of these TROSY-HSQC spectra further suggest it will bepossible to determine complete three-dimensional structures of suchintegral membrane proteins in micelles without protein purification.

These HSQC or TROSY-HSQC spectra can also be used to identify detergentsto use in the purification of the target protein, for use in biochemicalstudies, antibody production, NMR studies, and/or for crystallizationand three-dimensional structure determination by X-ray crystallography.

Selective Peptide Bond Labeling With SPP for High Throughput ResonanceAssignments

Selective labeling of the peptide bond between amino acid residue X andamino acid residue Y (where each of X and Y are any of the 20 common Lamino acids) in the sequence X-Y is provided by enriching the proteinwith amino acid X that is ¹³C-enriched in its backbone carbonyl positionand amino acid Y that is ¹⁵N-enriched in its backbone ¹⁵N atom. Thus, ina protein sequence, only the X-Y dipeptide sites will have ¹³C-¹⁵Nlabeled peptide bonds. These sites can be selectively observed usingHNCO type triple resonance NMR experiments, providing site-specificresonance assignments of the ¹⁵N, ¹³0, and ¹H atoms of the peptide bond,as well as other scalar-coupled or dipolar-coupled atoms. This PeptideBond Labeling (PBL) approach has been demonstrated using classical E.coli expression as well as using cell-free protein expression systems.

SPP-NMR with condensed fermentation is ideally suited for the PBLapproach. This method may be referred to as Single ProteinProduction-Peptide Bond Labeling (hereinafter “SPP-PBL”).

Using a robotic protein sample preparation platform (Acton et al, 2005)SPP-PBL can also be implemented in 96-well format. Using this approach,a series of 96 protein samples can be generated, each with a differentpeptide bond labeled. HNCO (or other NMR experiments) can be recorded onas little as 10 micrograms of these protein samples using 1 mm microNMRprobes and a robotic sample changer. In this way, backbone resonanceassignments can be obtained for 96 distinct sites in the proteinstructure.

This SPP-PBL technology is valuable for determining backbone resonanceassignments in soluble or membrane proteins, and particularly inperdeuterated membrane proteins. The information provided is valuablefor drug design and structure-function studies of proteins.

EXAMPLES Example 1

¹⁵N-¹H HSQC spectra was measured with non-purified, ¹⁵N-labeled RAMP-4in (A) 10% (vol/vol) bddm (B) 5% (vol/vol) bddm (C) 10% (vol/vol) Idao(D) 10% (vol/vol) zwittergent and (E) 10% DPC (vol/vol) micelles. Allthe spectra were collected on 800 MHz Bruker US2 spectrometer withcryoprobe at 20 degrees. The samples were prepared in the buffer of 10mM sodium phosphate, 75 mM sodium chloride, 50 μM EDTA, 5% D2O at pH7.5. The protein concentration was 200 fLM. The spectra with differentdetergents were recorded and processed in the same manner. The measuringtime for each spectrum was four and half hours. See FIG. 3.

Example 2

¹⁵N-¹H TROSY spectra measured with non-purified, ¹⁵N-labeled RAMP-4 in(A) 10% (vol/vol) bddm (B) 5% (vol/vol) bddm (C) 10% (vol/vol) Idao (D)10% (vol/vol) zwittergent and (E) 10% DPC (vol/vol) micelles. Samplepreparations and the NMR instrument used for data collection were thesame as in Example 1. The spectra with different detergent conditionswere recorded and processed identically. The data collection time foreach spectrum was nine and half hours. See FIG. 4.

1.-27. (canceled)
 28. A method of producing an isotope-labeled proteincomprising: i. contacting a vector comprising a gene encoding anmRNA-specific endoribonuclease and a gene encoding a target protein witha composition suitable to induce the mRNA-specific endoribonuclease; ii.contacting the vector with a selected isotope-labeled media to label,the target protein; and iii. contacting the vector with a compositionsuitable for inducing, the target protein to produce an isotope-labeledprotein, wherein each step is performed sequentially.
 29. The method ofclaim 28, wherein the isotope-labeled protein is utilized in a roboticexpression and purification system to determine protein resonanceassignments.
 30. The method of claim 28, wherein the isotope-labeledprotein is utilized in an automated NMR analysis to analyze proteinresonance assignments.
 31. The method of claim 28, wherein theisotope-labeled protein is utilized in HSQC or TROSY-HSQC methods toscreen detergent.
 32. The method of claim 31, wherein the screeneddetergent is utilized to identify suitable conditions for membraneprotein purification.
 33. The method of claim 28, wherein theisotope-labeled protein is utilized in NMR for resonance assignments.34. The method of claim 28, wherein the isotope-labeled protein isutilized in NMR for 3D structure analysis.